1. Technical Field
At least one example embodiment relates to a total internal reflection fluorescence microscope (TIRFM), and more particularly, to a TIRFM that may cause a stimulated emission depletion (STED) phenomenon through a de-excitation light source to acquire a resolution beyond a diffraction limited feature in a horizontal direction and a vertical direction by including an aperture that allows a light emitted from an excitation light source for exciting a fluorescent substance and a light emitted from the de-excitation light source for de-exciting the fluorescent substance to be overlappingly incident to an object lens using a high refractive solid immersion lens (SIL) and adjusts an angle at which a total internal reflection occurs on the SIL due to the excitation light source.
2. Related Art
In the related art, an optical microscope has been used to image a specimen such as a bio-sample. However, an optical microscope as shown in FIG. 1A has an issue in that a resolution is relatively low in a vertical direction.
A total internal reflection fluorescence microscope (TIRFM) as shown in FIG. 1B is a microscope using an evanescent wave that occurs due to total internal reflection. The TIRFM refers to a fluorescent imaging scheme by an evanescent wave occurring due to total internal reflection by allowing a light to be incident to a contact interface between a high refractive index medium and a low refractive index medium at a threshold angle or more.
In general, the TIRFM uses an oil immersion lens. In this case, a transmission depth of the evanescent wave is 100 nm or less, which is significantly less than a transmission depth of a general optical system. Accordingly, the TIRFM may acquire a further high resolution in a vertical direction (hereinafter, also referred to as a vertical resolution).
In particular, since the TIRFM is the fluorescent imaging scheme by the evanescent wave, the TIRFM may be easily applicable to image the surface of a specimen. Accordingly, the TIRFM may be utilized to many researches, such as membrane dynamics, a single module imaging field, etc., using a relatively high vertical resolution and a surface imaging characteristic.
While the TIRFM may increase a vertical resolution, a resolution in a horizontal direction (hereinafter, also referred to as a horizontal resolution) may be limited by a diffraction limited feature. In particular, due to a characteristic of the oil immersion lens that a refraction index of an immersive material is limited, a numerical aperture (NA) of the lens is limited to 1.49. Thus, a horizontal resolution is slightly less than a vertical resolution.
Referring to FIGS. 2 and 3, a stimulated emission depletion (STED) microscopy that is one of ultra-high resolution technologies according to the related art relates to a scheme of overlappingly emitting two lights of different modes to a specimen to be observed. Once a fist light is emitted toward the specimen, energy is absorbed at the specimen and fluorescence is generated. Here, if a second light in a donut shape having a nanometer-sized empty center is emitted and overlaps the first light, the fluorescence is observed at the central space with the fluorescence being prohibited in a remaining area excluding the central space. A nanometer-unit image may be acquired by minutely moving the light and emitting the light toward the overall specimen in the above manner. If many images acquired as above are merged into a single image, a final image may be configured to have a resolution less than 0.2 μm.